Figure 1. Impaired hippocampus-dependent contextual fear memory in constitutive Fmr1 KO and CA1-Fmr1 KO mice.

(A and B) Contextual fear memory test in constitutive Fmr1 KO mice. Freezing levels were measured immediately after fear conditioning training (A) and again in training context three days after (B) (***p<0.001, two-tailed unpaired t test). (C–D) Passive avoidance test in constitutive Fmr1 KO mice. (C) Learning curves (left) and trials number to criterion (right) were measured during training, and contextual fear memory in passive avoidance was measured 1 day later (D) (**p<0.01, two-tailed Mann-Whitney U test). (E) Representative images of hippocampal CA1 regions from Fmr1 conditional KO mice with bilateral injections of AAVs expressing Cre-GFP or mCre-GFP (green). FMRP expression was evaluated with immunostaining (red). Scale bars: 100 μm. (F and G) Contextual fear memory test in CA1-Fmr1 KO mice. Freezing levels were measured immediately after fear conditioning training (F) and again in training context 1 day after (G) (***p<0.001, two-tailed unpaired t test). n/N, number of mice/number of independent litters. All graphs represent mean ± SEM.

Figure 1—figure supplement 1. Behavioral characterization of constitutive Fmr1 KO mice and CA1-Fmr1 KO mice reared in home cage.

(A) Learning curves of constitutive Fmr1 KO mice in contextual fear conditioning. The intensive fear conditioning training paradigm has three sessions, each consists of four pairs of co-terminating tone (20 s, gray bar) and foot shock (2 s, red bar). Freezing levels before, during and after each tone-shock pair were quantified (Two-way repeated measure ANOVA: training 1: group factor, F(1, 16)=0.8305, p=0.3756; interaction, F(24, 384)=0.4943, p=0.9797; training 2: group factor, F(1, 16)=9.416, **p<0.01; interaction, F(24, 384)=1.170, p=0.2658; training 3: group factor, F(1, 16)=5.896, *p<0.05; interaction, F(24, 384)=1.959, **p<0.01). (B) Hot plate test. Latencies to escape were quantified (p=0.82, two-tailed Mann Whitney U test). (C) Y maze test. Percentage of spontaneous alterations was quantified (p=0.637, two-tailed unpaired t test). (D) Open field test. Left, quantification of track length in 30 min (p=0.1095, two-tailed unpaired t test); Right, quantification of percent track length in center (p=0.8692, two-tailed unpaired t test). (E) Elevated plus maze. Time spent on the open vs. closed arms was measured (Two-way ANOVA: group factor, F(1, 36)=0.5884, p=0.4481; interaction, F(1, 36)=1.853, p=0.1819). (F) Novel object recognition. Recognition index is defined as the ratio between time spent on novel object and time spent on both objects (p=0.9856, two-tailed unpaired t test). (G) A representative image of a hippocampal tissue section showing CA1-specific AAV-mediated expression of Syn-Cre-GFP. Scale bar: 200 μm. (H) Learning curves of CA1-Fmr1 KO mice in conventional contextual fear conditioning test. Three pairs of tone (30 s, gray bar) and foot shock (2 s, red bar) were delivered with a 60 s interval. Two-way repeated measure ANOVA: group factor, F(1, 19)=0.2128, p=0.6498; interaction, F(11, 209)=0.3191, p=0.9812. (I) Hot plate test. Quantification of latencies to escape (p=0.9602, two-tailed unpaired t test). (J) Y maze test. Quantification of percent spontaneous alterations (p=0.1707, two-tailed Mann Whitney U test). (K) Open field test. Left, quantification of track length in 30 min (p=0.7037, two-tailed unpaired t test); Right, quantification of percent track length in center (p=0.9375, two-tailed unpaired t test). (L) Novel object recognition. Recognition index defined as the ration between time spent on novel object and time spent on both objects (p=0.973, two-tailed unpaired t test). n/N, number of mice/number of independent litters. All graphs represent mean ± SEM.